It will be clearly understood that, although a number of prior art publications are referred to herein to describe background information, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents.
Angiogenesis, the formation of new blood vessels from pre-existing vessels, plays a critical role in many physiological and pathologic processes including embryogenesis, wound healing, tumour growth and metastasis (Augustin, 1998; Hanahan, 1997). Thus, the angiogenic process is considered an excellent target for therapeutic intervention. Identification of key regulatory molecules has principally used in vitro models in which endothelial cells (EC) are cultured on extracellular matrix (ECM) components such as collagen, fibrinogen or fibronectin, with identification of targets that are involved in events such as migration or proliferation which are elements of angiogenesis but are not specific for it. However, one problem in these investigations is the fact that the assays are generally performed on a flat or two dimensional (2D) environment, whereas EC morphogenesis to form capillary tubes requires a 3D matrix, allowing the establishment of important polarity cues. The use of such 3D assays, which include matrices of collagen type 1, fibrin, or Matrigel, recapitulates many of the events in angiogenesis, and has allowed dissection of the cellular and molecular events in angiogenesis (Gamble et al., 1993; 1999; Bayless and Davis, 2002; Meyer et al., 1997). Data using these assays to define genes altered during angiogenesis has supported the ideas firstly that there are fundamental differences between cells responding on 3D versus 2D matrices, and secondly that genes specific for angiogenesis might exist.
The mammalian Rho family of small GTPases has been implicated in diverse cellular functions, including reorganisation of the actin cytoskeleton, cell growth control, transcription regulation and membrane trafficking (Van Aelst and D'Souza-Schorey, 1997). The Rho family of small GTPases consists of at least 20 members: Rho (A,B,C), Rac (1,2,3), Cdc42, TC10, TCL, Chp (1,2), RhoG, Rnd (1,2,3), RhoBTB (1,2), RhoD, Rif and TTF (Etienne-Manneville and Hall, 2002). Like other members of the Ras superfamily, Rho proteins act as molecular switches to control cellular processes by cycling between active GTP-bound and inactive GDP-bound states. Regulation of these GTPases occurs via three major classes of regulatory proteins. The guanine nucleotide exchange factors (GEF) regulate activation through GDP-GTP exchange, GTPase-activating proteins (GAPs), which promote hydrolysis of the GTP to GDP-bound form, since the Rho proteins themselves display little if any basal GTPase activity and guanine nucleotide dissociation inhibitors (GDIs) which stabilise the inactive GDP-bound form of the protein (Mackay and Hall, 1998). At least 134 of these regulatory proteins have now been defined (Etienne-Manneville and Hall, 2002).
The function of the Rho family in endothelial morphogenesis is only now being elucidated, and it appears that different Rho family members play specific roles. Rho and Rac are important for regulation of permeability and cell migration (Wojciak-Stothard et al., 2001; Nobes and Hall, 1999). Rho is also important for EC attachment and apoptosis (Chrzanowska-Wodnicka and Burridge, 1996; Hippenstiel et al., 2002), while Cdc42 and Racl are implicated in vacuole and subsequent lumen formation (Bayless and Davis, 2002). Given the limited number of RhoGTPases and the seeming over-abundance of RhoGAPs, it is likely that the RhoGAPs may partly provide the specificity in control of function of the RhoGTPases.